Today 2.6 billion people live in areas of risk for Plasmodium vivax transmission; 106 - 313 million cases of vivax malaria annually. While Duffy blood group negativity has been considered to confer resistance to vivax malaria, our recent findings indicate that P. vivax has gained the capacity to infect erythrocytes and cause disease in Duffy-negative (Duffy(-)) people in Madagascar and suggests that P. vivax has acquire new erythrocyte infection mechanisms. In surveys of school-aged children 8.8% of asymptomatic Duffy(-) children (42/476) were P. vivax PCR-positive. Additionally, during surveys to assess in vivo efficacy of drugs recommended by the Madagascar Ministry of Health to treat malarial illness, we identified nine Duffy(-) people who had mono-infection P. vivax malaria (4.9% of 183 participants). We validated this unusual finding by microscopy to provide the first evidence for blood- stage development of P. vivax, including sexual-stage gametocytes necessary to continue the parasite lifecycle through mosquito transmission. Finally, flow cytometric analysis of erythrocytes from Malagasy study participants (n=43; many of whom experienced clinical P. vivax malaria) showed that Duffy(-) genotype and phenotype were 100% concordant, to indicate that the Duffy antigen was not cryptically expressed on the erythrocyte surface in genotypically Duffy(-) individuals. To begin understanding how P. vivax has gained capacity to infect Duffy(-) erythrocytes we propose to analyze the genome sequence of several P. vivax isolates extracted from blood samples of Duffy(+) and Duffy(-) Malagasy individuals, and the resulting data will allow us to address the following Specific Aims. Aim 1: Determine whether the same strains of P. vivax are responsible for infections of Duffy(-) and Duffy(+) individuals in Madagascar and reconstruct the population history of Malagasy P. vivax. Aim 2: Identify genes under recent positive selection in Malagasy P. vivax genomes. Our study of genetic diversity among multiple Malagasy P. vivax isolates by whole genome sequencing will allow us to determine how, or if the parasite population has partitioned in response to the important barrier imposed by Duffy negativity. By identifying loci under recent positive selection, our studies have strong potential to identify new parasite invasion ligands contributing to Duffy-independent erythrocyte invasion. PUBLIC HEALTH RELEVANCE:- Today 2.6 billion people live in areas of risk for Plasmodium vivax (Pv) transmission; 106 - 313 million cases of vivax malaria annually. Duffy blood group negativity has been considered to confer resistance to vivax malaria, however, our recent findings indicate that Pv has gained the capacity to infect erythrocytes and cause disease in Duffy-negative people in Madagascar, suggests that Pv has acquire new erythrocyte infection mechanisms. Through whole genome sequence analysis we will reveal differences between Pv strains infecting Duffy-negative vs. Duffy-positive people that will help identify genes evolving to expand the erythrocyte invasion capacity of Pv.